Putative T helper (Th)l-mediated autoimmune diseases such as multiple sclerosis (MS) and rheumatoid arthritis (RA) improve during late pregnancy when estrogen levels are high. In animal models of MS and RA, pregnancy doses of estrogens (estriol and estradiol) have been shown to ameliorate disease. In murine experimental autoimmune encephalomyelitis (EAE), estrogen treatment has been shown to cause a favorable shift in cytokine profile with an increase in production of the anti-inflammatory cytokine interleukin (IL)-10 during autoantigen-specific responses. The purpose of this application is to investigate further the mechanisms which underlie the protective effect of treatment of female EAE mice with pregnancy doses of estrogens (estriol and estradiol). In Specific Aim 1, it will be determined whether estrogens ameliorate disease by acting through estrogen receptor alpha or beta. This will be achieved by inducing EAE in ER beta knockout mice and assessing whether treatment with estrogens are still protective. The role of ER alpha will be determined by using a potent ER blocking agent (ICI 182,780) in ER beta knockout mice to ascertain whether the protective effects of estrogens are blocked when ER alpha is blocked. Treatment of EAE mice with selective ER alpha or beta agonists will confirm data above regarding whether estrogens act on ER alpha or beta when mediating disease protection. In Specific Aim 2, the immune cells that express each ER and the cells that are producing increased amounts of IL-10 during estrogen treatment will be identified. These studies will reveal whether estrogens increase IL-10 production by acting directly on a given immune cell versus acting indirectly via other immune cells. While an estrogen-mediated increase in IL-10 production by immune cells would be expected to be protective in EAE, this does not preclude additional protective mechanisms through which estrogens may act. Therefore, in Specific Aim 3, the possibility that estrogens may act through mechanisms other than increasing IL-10 production in immune cells will be examined. EAE will be induced in estrogen treated, IL-10 knockout mice to determine whether estrogen treatment is still protective in the absence of the IL-10 gene. Finally, the site of action of estrogens during EAE may be in the peripheral immune system and/or within the central nervous system (CNS). Systemic estrogen treatment used in combination with an ER blocking agent that acts only in the periphery will demonstrate whether estrogens act in the peripheral immune system or within the CNS. Administration of estrogens intra-ventricularly, within the CNS, will be used as a complementary approach. A detailed understanding of how each estrogen abrogates EAE pathogenesis may lead to treatment of MS and possibly other Th1-mediated autoimmune diseases with Selective Estrogen Receptor Modulators (SERMS) targeted to the pathogenic site of action.